DNA encoding mammalian neuropeptide FF (NPFF) receptors and uses thereof

Abstract

This invention provides isolated nucleic acids encoding mammalian NPFF receptors, purified mammalian NPFF receptors, vectors comprising nucleic acid encoding mammalian NPFF receptors, cells comprising such vectors, antibodies directed to mammalian NPFF receptors, nucleic acid probes useful for detecting nucleic acid encoding mammalian NPFF receptors, antisense oligonucleotides complementary to unique sequences of nucleic acid encoding mammalian NPFF receptors, transgenic, nonhuman animals which express DNA encoding normal or mutant mammalian NPFF receptors, methods of isolating mammalian NPFF receptors, methods of treating an abnormality that is linked to the activity of the mammalian NPFF receptors, as well as methods of determining binding of compounds to mammalian NPFF receptors, methods of identifying agonists and antagonists of NPFF receptors, and agonists and antagonists so identified.

What is claimed is:  
     
       1. A process involving competitive binding for identifying a chemical compound which specifically binds to a human NPFF2 receptor which comprises separately contacting cells expressing on their cell surface the human NPFF2 receptor, wherein such cells do not normally express the human NPFF2 receptor, with both the chemical compound and a second chemical compound known to bind to the receptor, and with only the second chemical compound, under conditions suitable for binding of both compounds, and detecting specific binding of the chemical compound to the human NPFF2 receptor, a decrease in the binding of the second chemical compound to the human NPFF2 receptor in the presence of the chemical compound indicating that the chemical compound binds to the human NPFF2 receptor, wherein the human NPFF2 receptor has an amino acid sequence identical to the amino acid sequence shown in FIG. 8 (SEO ID NO: 6) or that encoded by plasmid pCDNA3.1-hNPFF2b (ATCC Accession No. 203255). 
     
     
       2. A process involving competitive binding for identifying a chemical compound which specifically binds to a human NPFF2 receptor which comprises separately contacting a membrane preparation from cells expressing on their cell surface the human NPFF2 receptor, wherein such cells do not normally express the human NPFF2 receptor, with both the chemical compound and a second chemical compound known to bind to the receptor, and with only the second chemical compound, under conditions suitable for binding of both compounds, and detecting specific binding of the chemical compound to the human NPFF2 receptor, a decrease in the binding of the second chemical compound to the human NPFF2 receptor in the presence of the chemical compound indicating that the chemical compound binds to the human NPFF2 receptor, wherein the human NPFF2 receptor has an amino acid sequence identical to the amino acid sequence shown in FIG. 8 (SEO ID NO: 6) or that encoded by plasmid pCDNA3.1-hNPFF2b (ATCC Accession No. 203255). 
     
     
       3. The process of  claim 1  or  2 , wherein the cell is an insect cell. 
     
     
       4. The process of  claim 1  or  2 , wherein the cell is a mammalian cell. 
     
     
       5. The process of  claim 4 , wherein the cell is nonneuronal in origin. 
     
     
       6. The process of  claim 5 , wherein the nonneuronal cell is a COS-7 cell, 293 human embryonic kidney cell, a CHO cell, a NIH-3T3 cell, a mouse Y1 cell, or a LM(tk−) cell. 
     
     
       7. The process of  claim 6 , wherein the compound is not previously known to bind to a human NPFF2 receptor. 
     
     
       8. A method of screening a plurality of chemical compounds not known to bind to a human NPFF2 receptor to identify a compound which specifically binds to the human NPFF2 receptor, which comprises 
       (a) contacting cells transfected with and expressing DNA encoding the human NPFF2 receptor with a compound known to bind specifically to the human NPFF2 receptor;  
       (b) contacting the cells of step (a) with the plurality of compounds not known to bind specifically to the human NPFF2 receptor, under conditions permitting binding of compounds known to bind to the human NPFF2 receptor;  
       (c) determining whether the binding of the compound known to bind to the human NPFF2 receptor is reduced in the presence of any compound within the plurality of compounds, relative to the binding of the compound in the absence of the plurality of compounds; and if so  
       (d) separately determining the binding to the human NPFF2 receptor of compounds included in the plurality of compounds, so as to thereby identify the compound which specifically binds to the human NPFF2 receptor;  
       wherein the human NPFF2 receptor has an amino acid sequence identical to the amino acid sequence shown in FIG. 8 (SEO ID NO: 6) or that encoded by plasmid pCDNA3.1-hNPFF2b (ATCC Accession No. 203255). 
     
     
       9. A method of screening a plurality of chemical compounds not known to bind to a human NPFF2 receptor to identify a compound which specifically binds to the human NPFF2 receptor, which comprises 
       (a) contacting a membrane preparation from cells transfected with and expressing DNA encoding the human NPFF2 receptor with the plurality of compounds not known to bind specifically to the human NPFF2 receptor under conditions permitting binding of compounds known to bind to the human NPFF2 receptor;  
       (b) determining whether the binding of a compound known to bind to the human NPFF2 receptor is reduced in the presence of any compound within the plurality of compounds, relative to the binding of the compound in the absence of the plurality of compounds; and if so  
       (c) separately determining the binding to the human NPFF2 receptor of compounds included in the plurality of compounds, so as to thereby identify the compound which specifically binds to the human NPFF2 receptor;  
       wherein the human NPFF2 receptor has an amino acid sequence identical to the amino acid sequence shown in FIG. 8 (SEQ ID NO: 6) or that encoded by plasmid QCDNA3.1-hNPFF2b (ATCC Accession No. 203255). 
     
     
       10. A method of  claim 8  or  9 , wherein the cell is a mammalian cell. 
     
     
       11. A method of  claim 10 , wherein the mammalian cell is non-neuronal in origin. 
     
     
       12. The method of  claim 11 , wherein the non-neuronal cell is a COS-7 cell, a 293 human embryonic kidney cell, a LM(tk−) cell, a CHO cell, a mouse Y1 cell, or an NIH-3T3 cell. 
     
     
       13. A process for determining whether a chemical compound specifically binds to and inhibits activation of a human NPFF2 receptor, which comprises separately contacting cells producing a second messenger response and expressing on their cell surface the human NPFF2 receptor, wherein such cells do not normally express the human NPFF2 receptor, with both the chemical compound and a second chemical compound known to activate the human NPFF2 receptor, and with only the second chemical compound, under conditions suitable for activation of the human NPFF2 receptor, and measuring the second messenger response in the presence of only the second chemical compound and in the presence of both the second chemical compound and the chemical compound, a smaller change in the second messenger response in the presence of both the chemical compound and the second chemical compound than in the presence of only the second chemical compound indicating that the chemical compound inhibits activation of the human NPFF2 receptor, wherein the human NPFF2 receptor has an amino acid sequence identical to the amino acid sequence shown in FIG. 8 (SEQ ID NO: 6) or that encoded by plasmid pCDNA3.1-hNPFF2b (ATCC Accession No. 203255). 
     
     
       14. The process of  claim 13 , wherein the second messenger response comprises chloride channel activation and the change in second messenger response is a smaller increase in the level of inward chloride current in the presence of both the chemical compound and the second chemical compound than in the presence of only the second chemical compound. 
     
     
       15. The process of  claim 13  or  14 , wherein the cell is an insect cell. 
     
     
       16. The process of  claim 13  or  14 , wherein the cell is a mammalian cell. 
     
     
       17. The process of  claim 16 , wherein the mammalian cell is nonneuronal in origin. 
     
     
       18. The process of  claim 17 , wherein the nonneuronal cell is a COS-7 cell, CHO cell, 293 human embryonic kidney cell, NIH-3T3 cell or LM(tk−) cell. 
     
     
       19. The process of  claim 13  or  14 , wherein the compound is not previously known to bind to a human NPFF2 receptor. 
     
     
       20. A method of screening a plurality of chemical compounds not known to inhibit the activation of a human NPFF2 receptor to identify a compound which inhibits the activation of the human NPFF2 receptor, which comprises: 
       (a) contacting cells transfected with and expressing the human NPFF2 receptor with the plurality of compounds in the presence of a known human NPFF2 receptor agonist, under conditions permitting activation of the human NPFF2 receptor;  
       (b) determining whether the activation of the human NPFF2 receptor is reduced in the presence of the plurality of compounds, relative to the activation of the human NPFF2 receptor in the absence of the plurality of compounds; and if so  
       (c) separately determining the inhibition of activation of the human NPFF2 receptor for each compound included in the plurality of compounds, so as to thereby identify the compound which inhibits the activation of the human NPFF2 receptor;  
       wherein the human NPFF2 receptor has an amino acid sequence identical to the amino acid sequence shown in FIG. 8 (SEQ ID NO: 6) or that encoded by plasmid pCDNA3.1-hNPFF2b (ATCC Accession No. 203255). 
     
     
       21. A method of  claim 20 , wherein the cell is a mammalian cell. 
     
     
       22. A method of  claim 21 , wherein the mammalian cell is non-neuronal in origin. 
     
     
       23. The method of  claim 22 , wherein the non-neuronal cell is a COS-7 cell, a 293 human embryonic kidney cell, a LM(tk−) cell or an NIH-3T3 cell.